Arabidopsis RIBA proteins: Two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis

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Abstract

Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interestingly, both AtRIBA2 and AtRIBA3 have amino acid exchanges in conserved peptides domains that have been found to be essential for the two enzymatic functions. In vitro activity assays of GCHII and DHBPS with all of the three purified recombinant AtRIBA proteins and complementation of E. coli ribA and ribB mutants lacking DHBPS and GCHII expression, respectively, confirmed the loss of bifunctionality for AtRIBA2 and AtRIBA3. Phylogenetic analyses imply that the monofunctional, bipartite RIBA3 proteins, which have lost DHBPS activity, evolved early in tracheophyte evolution.© 2012 by the authors; licensee MDPI, Basel, Switzerland.

Figures

  • Figure 1. Biosynthesis of riboflavin. Riboflavin biosynthesis is initiated by the enzymes GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) converting GTP (1) into 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (2) and ribulose-5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphate (9), respectively. Both 2 and 9 are the first committed substrates of the riboflavin biosynthetic pathway. Following the biosynthetic pathway to riboflavin (7), 2 is consecutively modified to 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidine 5'-phosphate (3), 5-amino-6ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (4) and 5-amino-6-ribitylamino2,4(1H,3H)-pyrimidinedione (5) by deaminase, reductase and phosphatase reactions, respectively. At present, it is still not clear if one specific phosphatase or several less specific enzymes are implemented in the dephosphorylation of 4. 5 is condensed with 9 by the enzyme lumazine synthase giving rise to 6,7-dimethyl-8-ribityllumazine (6). Finally, riboflavin synthase catalyzes a dismutation reaction of two molecules of 6 to form 7 yielding 5 as a byproduct, which again serves as substrate for lumazine synthase.
  • Figure 2. qPCR analyses of AtRIBA genes. (A) transcript accumulation of Arabidopsis RIBA genes was examined in different tissue types and (B), in two representative AtRibA1 antisense lines with intermediate (A1#5) and strong bleaching phenotype (A1#2), respectively. Expression was calculated relative to mRNA levels of SAND (At2g28390).
  • Figure 3. Phenotype of antisense AtRIBA1 plants. (A) Different degrees of bleaching are the result of AtRIBA1 antisense expression. Plants displaying a moderate antisense phenotype (left panel) start to bleach partially at the tip of leaves in the rosette stage, while individuals with a stronger reduction in AtRIBA1 transcript amounts display white inner rosette leaves and shoot apical meristem (middle). A Arabidopsis thaliana ecotype Columbia (Col-0) wild-type plant of the same age is depicted in the right panel. (B) For HPLC analyses, leaves of line A1#2 were harvested and dissected as indicated. I: green, II: medium, III: white pigmentation. Reference samples were collected from comparable regions of wild-type plants. (C) Leaf regions depicted in (B) were subjected to flavin extraction and analyzed for the content of riboflavin using HPLC. (D) Immunodetection of AtRIBA protein in whole leaf extracts of line A1#2 and wild-type Col-0 control using anti-RIBA1 specific antiserum. Both samples represent identical fresh weight amounts. Although the antiserum recognizes all three AtRIBA isoforms, the upper band (arrow head) was shown to represent AtRIBA1 by an analysis of overexpressing lines (data not shown).
  • Figure 4. Green fluorescent protein (GFP) localization experiments. (A) The amino terminal sequences comprising the putative transit peptides were translationally fused to the N-terminus of GFP. Targeting properties were predicted using TargetP and Predotar. The lengths of the AtRIBA aminotermini tested experimentally are indicated. (B) RIBA-GFP fusions were expressed transiently in Agrobacterium-infiltrated Nicotiana benthamiana leaves and visualized in mesophyll cells using Confocal Laser Scanning Microscopy. The three panels show merged images of GFP and chlorophyll fluorescence indicating that green fluorescence localizes within the plastid compartment for all three RIBA-GFP fusion constructs investigated.
  • Figure 5. Enzymatic activities of AtRIBA proteins. (A–C) His-tagged N-terminally truncated AtRIBA proteins were overexpressed in E. coli and purified by FPLC. (A) Coomassie staining following SDS PAGE detects highly enriched recombinant Arabidopsis proteins in selected FPLC fractions. 0.75 μg of each recombinant RIBA protein were applied. The obtained fractions were assayed in vitro for enzymatic activity for GCHII (B) and DHBPS (C). In both assays a standard RIBA protein (RibA from Bacillus subtilis) was included as positive control. (D,E) E. coli ribA (D) and ribB (E) mutants were transformed with plasmids encoding the three Arabidopsis RIBA isoforms. Growth (OD600) of at least three independent cultures in liquid M9 minimal medium was monitored for 24 h; the initial absorption of the culture was subtracted. Empty vector pACYC184 was used as negative control; the maximum density reached by the control did not exceed an OD600 of 0.2 (grey line). Standard errors are indicated.
  • Figure 6. Phylogenetic tree of selected plant RIBA protein sequences. RIBA gene families identified in the angiosperm species Arabidopsis thaliana (At), Vitis vinifera (Vv) and Oryza sativa (Os), two RIBA proteins from a lycopodiophyte species (Selaginella, Sm), as well as single RIBA sequences from a moss (Physcomitrella, Pp), a green algae (Chlamydomonas, Cr), and a cyanobacterium (Synechococcus, Syn) are included in the analysis. Alignment (using MUSCLE 3.7 and Gblocks 0.91b), phylogenetic analysis (PhyML3.0 aLRT) and tree rendering (TreeDyn 198.3) were performed using the phylogeny resource (http://www.phylogeny.fr) [29], the tree was re-rooted using SynRIBA as outgroup. Full length RIBA amino acid sequences of the following species were used: Arabidopsis thaliana (accession nrs. NP_201235, NP_179831, NP_568913), Chlamydomonas reinhardtii (XP_001689850), Oryza sativa (NP_001047195, BAD09287, NP_001055757), Physcomitrella patens (XP_001770447), Selaginella moellendorfii (XP_002962016, XP_002960875), Synechococcus sp. PCC 7002 (YP_001733693), Vitis vinifera (XP_002267374, XP_002266093, XP_002281446).

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APA

Hiltunen, H. M., Illarionov, B., Hedtke, B., Fischer, M., & Grimm, B. (2012). Arabidopsis RIBA proteins: Two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis. International Journal of Molecular Sciences, 13(11), 14086–14105. https://doi.org/10.3390/ijms131114086

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